An essential step of the mitotic process is the attachment of chromosomes to the spindle by kinetochore microtubules. The fact that both attachment and the formation of kinetochore microtubules occur during prometaphase has led to speculation that the mechanisms underlying these processes may be coupled. The research outlined in this proposal is designed to improve our understanding of spindle ultrastructure during prometaphase and thereby may clarify the relationship between the formation of kinetochore microtubules and attachment. Several experiments are planned to test whether kinetochore microtubules are made by kinetochore-initiated assembly or by recruitment of preformed microtubules that become kinetochore microtubules as a result of their attachment to kinetochores. Suggested approaches include: (a) the use of mitotic poisons, such as Colcemid, to test the effect of inhibition of microtubule assembly on kinetochore microtubule formation, (b) lysed cells to test both the recruitment and assembly capacities of kinetochores, and (c) microbeam irradiation with 366 nm light to inactivate Colcemid inhibition and determine the growth zones of microtubules in the spindle. The results may be significant with regard to the role of the kinetochore in making kinetochore microtubules and in bringing about the attachment of chromosomes to the spindle.